Attenuated Pasteurella multocida vaccines and methods of making and use thereof

ABSTRACT

The present invention provides attenuated  P. multocida  strains that elicit an immune response in animal  P. multocida , compositions comprising said strains, methods of vaccination against  P. multocida , and kits for use with such methods and compositions. The invention further provides novel, genetically-engineered mutations in  P. multocida  hyaD and nanPU genes, which are useful in the production of novel attenuated  P. multocida  bacterial strains.

This application claims priority to provisional application U.S. Ser.No. 61/898,497, filed on Nov. 1, 2013, and incorporated by referenceherein in its entirety. All references cited herein, are incorporated byreference herein, in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to attenuated bacterialvaccines, particularly those providing broad, safe, and effectiveprotection to bovines against infections/disease caused by PasteurellaMultocida. The invention further relates to methods of producing theattenuated bacteria, and to the identification of nucleic acidvariations that are associated with decreased virulence of theattenuated bacteria.

The invention accordingly relates to immunogenic or vaccine compositionscomprising the bacteria of the invention; e.g., live attenuatedbacteria. The bacteria also could be inactivated in the compositions;but it may be advantageous that the bacteria are live attenuated P.multocida bacteria. The invention therefore further relates to methodsfor preparing and/or formulating such compositions; e.g., culturing orgrowing or propagating the bacteria on or in suitable medium, harvestingthe bacteria, optionally inactivating the bacteria, and optionallyadmixing the bacteria with a suitable veterinarily or pharmaceuticallyacceptable carrier, excipient, diluent or vehicle and/or an adjuvantand/or stabilizer. Thus, the invention also relates to the use of thebacteria in formulating such compositions.

BACKGROUND OF THE INVENTION

Pasteurella multocida is a gram-negative, non-motile, rod shaped,facultative anaerobe which is isolated from a wide range of animals andbirds from all over the world.

The P. multocida isolates are classified into five serogroups (A, B, D,E and F) based on capsular antigens and 16 serotypes by somatic antigens(Rimler and Rhoades, 1989). Serogroup A is most commonly associated withfowl cholera in birds followed by serogroup D (Rhoades and Rimler,1989). Among the isolates, serogroup F strains are predominantlyisolated from poultry and turkeys, but rarely from calves (Shewen andConlon, 1993; Catry et al., 2005). In pigs, atrophic rhinitis andpneumonia are primarily associated with serogroups D and A which expressdermonecrotizing toxin (Dungworth, 1985). On the other hand P. multocidaserogroups B and E are usually associated with hemorrhagic septicemia incattle and water buffaloes in tropical and sub-tropical regions ofAfrica and Asia (Carter and de Alwis, 1989; Rimler and Rhoades, 1989;Shewen and Conlon, 1993). In contrast P. multocida serogroups B and Eare rarely isolated North America cattle population (Confer, 1993). Morethan 92% of P. multocida isolated from the US cattle which cause severesuppurative bronchopneumonia belong to serotype A:3 (Ewers et al., 2006;Confer et al., 1996; Weekley et al., 1998). P. multocida infection incalves results in significant production yield losses and mortality(Ewers et al., 2006; Confer et al., 1996; Dalgleish, 1989; Weekley etal., 1998). Furthermore, P. multocida is often associated with bovinerespiratory disease complex (BRDC) along with Mannheima haemolytica andHistophilus somni. From 2001, bovine pneumonic pasteurellosis due to P.multocida infection has increased in the UK cattle population. In manyUK cases, P. multocida infections exceeded the number of outbreakscaused by M. haemolytica induced bovine bacterial pneumonia (VeterinaryLaboratories Agency, 2007). Worldwide, P. multocida serogroup A isolatesare one of the major pathogens associated with BRDC (Frank, 1989; Rimlerand Rhoades, 1989).

P. multocida isolates associated with BRDC have numerous virulence orpotential virulence and virulence-associated factors like adhesins andfilamentous hemagglutinin which aid in adherence and colonization, ironacquisition proteins and transport systems, extracellular enzymes suchas neuraminidase, endotoxin (lipopolysaccharide, LPS), polysaccharidecapsule and a variety of outer membrane proteins (OMPs). Immunity ofcattle against respiratory pasteurellosis is poorly understood; howeversome reports indicate that high serum antibodies against P. multocidaOMPs are important for enhancing resistance against this bacterium.

There are a few commercial vaccines currently available against P.multocida for use in cattle. These vaccines are predominatelytraditional bacterins and a live streptomycin-dependent mutant. However,the field efficacy of these vaccines is questionable and none of thevaccines afford reliable protection. Therefore, there remains a need forsafe and effective vaccines to protect cattle against P. multocidainfections.

State of the Art Review

Intervet (Merck Animal Health) makes a hyaE gene-deleted P. multocidavaccine. In contrast, the instantly disclosed P. multocida vaccine ishyaD gene deletion mutant. HyaD is a different gene in the same locus,and although both the gene deletions result in an acapsular phenotype, askilled person could not have predicted ahead of this disclosure whetherdeleting the hyaD gene would result in a stable, viable acapsularphenotype. Moreover, according to U.S. Pat. No. 7,351,416 B2 (Examples 3& 4), the ΔhyaE vaccine may be administered to steers (weighing about500 pounds), or 2-3 month old calves (weighing over 150 pounds). Incontrast, the target animal for the vaccines of the instant disclosureare calves as young as 4-6 weeks old, and weighing significantly less.Immune responses of very young animals are significantly different thanolder ones. Furthermore, vaccine safety is of paramount importance whenused in young calves.

EP1831248B1 (to Intervet) describes a transposon generated mutant P.multocida, which is not directed or site specific. Bacteria harboringsuch transposon insertions are not likely to be approved by regulatoryagencies for use in vaccines, and so disclosure of these types ofmutations may fairly be viewed as preliminary work leading to targetedgene modification, including deletion. The mutant gene is reported as“ORF 15,” which is a membrane bound lysozyme inhibitor of c-typelysozyme. Finally, the vaccination challenge was done in poultry ratherthan calves.

WO2003086277A2 (to Merial) discloses attenuated P. multocida 1059. Thegene deletions were initially produced using random signature taggedmutagenesis using transposon Tn5. Along with many specific andnon-specific mutants, this library has mutants lacking PhyA, hyaC andhyaE genes which are involved with capsule biosynthesis. However, thesemutants were generated by random mutagenesis and their genetic stabilityhas not been tested over a long period. This is a critical property fora vaccine to be used as a modified live product under field conditions.Furthermore, P. multocida 1059 is an avian strain unsuitable for calfvaccination.

SUMMARY OF THE INVENTION

An object of this disclosure is to provide attenuated bacteria as wellas methods for treatment and prophylaxis of infection by P. multocida.

The present disclosure further relates to efficacious field vaccinescomprising attenuated P. multocida strains for use as vaccines incattle. Mutant strains according to the instant disclosure may exhibitreduced or no expression of hyaD, nanPU genes, or both. Moreover,methods of producing the attenuated bacteria, as well as methods forproviding cattle immunity, including protective immunity, againstsubsequent infections are disclosed herein. Kits comprising at least theattenuated P. multocida strain and instructions for use are alsoprovided.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF DRAWINGS

A full and enabling disclosure of the present invention, including thebest mode thereof, to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, includingreference to the accompanying figures, wherein:

FIG. 1A is part of a flow diagram (FIGS. 1A-1F) showing the constructionof the P. multocida 1062 hyaD mutant. FIG. 1A shows the insertion of thehyaD-containing fragment into the pCR2.1 vector;

FIG. 1B depicts the removal of the BglII fragment to produce theΔhyaD-containing pCR2.1 vector;

FIG. 1C depicts the shuttling of the ΔhyaD-containing fragment into thepBC SK-vector;

FIG. 1D depicts the insertion of the Tn903 Kan-containing SalI fragmentinto the ΔhyaD-containing pBC SK-vector;

FIG. 1E depicts the insertion of the pCT109GA189 ts on into the ΔhyaD-and Tn903 Kan-containing pBC SK-vector, to form the “replacement”vector;

FIG. 1F depicts the replacement of the genomic hyaD sequence with theΔhyaD sequence;

FIG. 2 is a gel image showing the hyaD-specific PCR products amplifiedfrom the P. multocida 1062 hyaDΔ-4PKL strain (lane 2) and the virulentparental P. multocida 1062 wild type strain (lane 3);

FIG. 3A is part of a flow diagram (FIGS. 3A-3F) showing the constructionof the P. multocida 1062 nanPU mutant. FIG. 1A shows the construction ofthe nanP/nanU fusion sequence-containing pCR2.1 vector from a fragmentcontaining the full-length nanP and nanU sequences;

FIG. 3B depicts the insertion of the nanP/nanU fusion sequence into thepBC SK-vector;

FIG. 3C depicts the insertion of the Tn903 Kan-containing SalI fragmentinto the nanPU fusion-containing pBC SK-vector;

FIG. 3D depicts the insertion of the pCT109GA189 ts on into the nanPUfusion- and Tn903 Kan-containing pBC SK-vector, to form the“replacement” vector;

FIG. 3E depicts the integration of the replacement plasmid into thechromosome;

FIG. 3F depicts the resolution of the replacement plasmid from thechromosome;

FIG. 4 is a gel image showing the nanPU-specific PCR products amplifiedfrom the P. multocida 1062 truncated nanPU strain (lane 2) and thevirulent parental P. multocida 1062 wild type strain (lane 3);

FIG. 5 is a gel image showing 1) P. multocida ΔnanPU/HyaD mutantamplified with nanPUF/nanPUR primers (1.3 Kb); 2) P. multocidaΔnanPU/HyaD mutant amplified with hyaDF/hyaDR primers (2.691 Kb); 3) P.multocida 1062 wild type amplified with nanPUF/nanPUR primers (3.150Kb); and 4) P. multocida 1062 wild type amplified with hyaDF/hyaDRprimers (2.916 Kb);

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleotide sequences and genes involvedin the attenuation of a microorganism, such as bacteria, for instance,Gram negative bacteria, e.g., Pasteurella multocida (P. multocida),products (e.g., proteins, antigens, immunogens, epitopes) encoded by thenucleotide sequences, methods for producing such nucleotide sequences,products, micro-organisms, and uses therefor, such as for preparingvaccine or immunogenic compositions or for eliciting an immunological orimmune response or as a vector, e.g., as an expression vector (forinstance, an in vitro or in vivo expression vector).

In order to develop an efficacious P. multocida field vaccine, threeattenuated strains were genetically engineered: 1) a hyaD partialdeletion mutant, which is unable to synthesize glycosyl transferase, andso exhibits the acapsular phenotype; 2) a nanPU deletion, which isunable to add sialic acid residues to terminal lipooligosaccharides; and3) a double knockout mutant lacking both hyaD and nanPU genes.

Mutations, including deletions and partial deletions, introduced intonucleotide sequences and genes of micro-organisms produce novel andnonobvious attenuated mutants. These mutants are useful for theproduction of live attenuated immunogenic compositions or liveattenuated vaccines having a high degree of immunogenicity.

These mutants are also useful as vectors which can be useful forexpression in vitro of expression products, as well as for reproductionor replication of nucleotide sequences (e.g., replication of DNA), andfor in vivo expression products.

Identification of the mutations provides novel and nonobvious nucleotidesequences and genes, as well as novel and nonobvious gene productsencoded by the nucleotide sequences and genes.

Such gene products provide antigens, immunogens and epitopes, and areuseful as isolated gene products.

Such isolated gene products, as well as epitopes thereof, are alsouseful for generating antibodies, which are useful in diagnosticapplications.

Such gene products, which can provide or generate epitopes, antigens orimmunogens, are also useful for immunogenic or immunologicalcompositions, as well as vaccines.

In an aspect, the invention provides bacteria containing an attenuatingmutation in a nucleotide sequence or a gene wherein the mutationmodifies, reduces or abolishes the expression and/or the biologicalactivity of a polypeptide or protein encoded by a gene, resulting inattenuated virulence of the bacterium.

The mutation need not be located within a coding sequence or gene todisrupt its function, leading to attenuation. The mutation can also bemade in nucleotide sequences involved in the regulation of theexpression of the gene, for instance, in regions that regulatetranscription initiation, translation and transcription termination.Thus also included are promoters and ribosome binding regions (ingeneral these regulatory elements lie approximately between 60 and 250nucleotides upstream of the start codon of the coding sequence or gene;Doree S M et al., J. Bacteriol. 2001, 183(6): 1983-9; Pandher K et al.,Infect. Imm. 1998, 66(12): 5613-9; Chung J Y et al., FEMS Microbiolletters 1998, 166: 289-296), transcription terminators (in general theterminator is located within approximately 50 nucleotides downstream ofthe stop codon of the coding sequence or gene; Ward C K et al., Infect.Imm. 1998, 66(7): 3326-36). In the case of an operon, such regulatoryregions may be located in a greater distance upstream of the gene orcoding sequence. A mutation in an intergenic region can also lead toattenuation.

A mutation within such regulatory sequences associated with the codingsequence or gene so that the mutation of this nucleotide sequencemodifies, inhibits or abolishes the expression and/or the biologicalactivity of the polypeptide or the protein encoded by the gene,resulting in attenuated virulence of the bacterium would be anequivalent to a mutation within a gene or coding sequence identified inthe present invention

Attenuation reduces or abolishes the pathogenicity of the bacteria andthe gravity of the clinical signs or lesions, decreases the growth rateof the bacteria, and prevents the death from the bacteria.

In particular, the present invention encompasses attenuated P. multocidastrains and vaccines comprising the same, which elicit an immunogenicresponse in an animal, particularly the attenuated P. multocida strainsthat elicit, induce or stimulate a response in a bovine.

Particular P. multocida attenuated strains of interest have mutations ingenes, relative to wild type virulent parent strain, which areassociated with virulence. It is recognized that, in addition to strainshaving the disclosed mutations, attenuated strains having any number ofmutations in the disclosed virulence genes can be used in the practiceof this invention.

In an embodiment, the attenuated strains comprise mutations in nucleicacid sequences comprising the nucleotides as set forth in SEQ ID NOs:1,5, or both. The attenuated strains may also comprise mutations insequences having at least 70% identity to the sequences as set forth inSEQ ID NOs:1, 5, or both, with the proviso that the homologous sequencesmust encode homologous proteins having comparable functions to thoseencoded by SEQ ID NO:1 or 5. Examples of comparable functions includethe ability to catalyze the same enzymatic reaction and the ability toserve the same structural role. The attenuated strains may also comprisemutations in sequences having at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identity to the sequences as set forth in SEQ ID NOs:1,5, or both, with the same proviso.

In an embodiment, the attenuated strains may comprise sequences havingat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to thesequences as set forth in SEQ ID NOs:11, 12, or both, provided that thehomologous sequences result in the attenuated phenotype, wherein theattenuated strains are comparably capable of safely eliciting an immuneresponse relative to attenuated strains comprising the sequences as setforth in SEQ ID NOs:11, 12, or both. The skilled person understands wellthat the attenuated strains comprise the mutated sequence(s) in place ofthe wild-type sequence(s). Thus, it is not intended that the inventionshould encompass, for example, a strain comprising both SEQ ID NO:1(wild-type) and SEQ ID NO:3 (mutated). However, the inventors doenvision that any deletion/modification of either or both SEQ ID NO:1and SEQ ID NO:5 may transform a virulent P. multocida strain into anattenuated strain, according to the instant disclosure.

In another embodiment, the attenuated P. multocida strains comprisenucleic acids encoding a peptide having the sequence as set forth in SEQID NOs:4, 13, or a peptide having 80%, 85%, 90%, 95%, or 98% homologythereto, and having comparable function thereto. In yet anotherembodiment, the strains comprise nucleic acids encoding peptides havingat least one amino acid substitution with respect to the sequences asset forth in SEQ ID NOs:4, 13, or both.

At the time of this disclosure, the mutants described herein were notknown to exist in any naturally-occurring P. multocida genomes, and wereonly produced as a result of the disclosed mutagenesis methods.

In yet another embodiment, the attenuated P. multocida strain hasmutations in the same genes, relative to its virulent parental strain,as the strain deposited at the American Type Culture Collection (ATCC®),in accordance with the Budapest Treaty, under the Patent DepositDesignation PTA-120624 (i.e. P. mult.1062 Nan May 9, 2012). ThePTA-120624 attenuated P. multocida strain was deposited on Oct. 16,2013, at the ATCC® depository located in Manassas, Va. (ie, 10801University Blvd, Manassas, Va. 20110), These mutations result in theattenuated strain having reduced virulence relative to it virulentparental strain.

In a particular embodiment, the attenuated strain is the straindeposited at the ATCC under the Patent Deposit Designation PTA-120624(i.e. P. mult.1062 Nan May 9, 2012).

In another aspect, the novel attenuated P. multocida strains areformulated into safe, effective vaccine against P. multocida andinfections/diseases cause by P. multocida.

In an embodiment, the P. multocida vaccines further comprise anadjuvant. In a particular embodiment, the adjuvant is a mucosaladjuvant, such as chitosan, methylated chitosan, trimethylated chitosan,or derivatives or combinations thereof.

In an embodiment, the adjuvant comprises whole bacteria and/or viruses,including H. parasuis, clostridium, swine influenza virus (SIV), bovinecircovirus (PCV), bovine reproductive and respiratory syndrome virus(PRRSV), Mannheimia, Pasteurella, Histophilus, Salmonella, Escherichiacoli, or combinations and/or variations thereof. In several embodiments,the adjuvant increases the animal's production of IgM, IgG, IgA, and/orcombinations thereof.

By “antigen” or “immunogen” means a substance that induces a specificimmune response in a host animal. The antigen may comprise a wholeorganism, killed, attenuated or live; a subunit or portion of anorganism; a recombinant vector containing an insert with immunogenicproperties; a piece or fragment of DNA capable of inducing an immuneresponse upon presentation to a host animal; a polypeptide, an epitope,a hapten, or any combination thereof. Alternately, the immunogen orantigen may comprise a toxin or antitoxin.

The terms “protein”, “peptide”, “polypeptide” and “polypeptide fragment”are used interchangeably herein to refer to polymers of amino acidresidues of any length. The polymer can be linear or branched, it maycomprise modified amino acids or amino acid analogs, and it may beinterrupted by chemical moieties other than amino acids. The terms alsoencompass an amino acid polymer that has been modified naturally or byintervention; for example disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling or bioactivecomponent.

The term “immunogenic or antigenic polypeptide” as used herein includespolypeptides that are immunologically active in the sense that onceadministered to the host, it is able to evoke an immune response of thehumoral and/or cellular type directed against the protein. Preferablythe protein fragment is such that it has substantially the sameimmunological activity as the total protein. Thus, a protein fragmentaccording to the invention comprises or consists essentially of orconsists of at least one epitope or antigenic determinant. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein, analogs thereof, or immunogenicfragments thereof. By “immunogenic fragment” is meant a fragment of aprotein which includes one or more epitopes and thus elicits theimmunological response described above. Such fragments can be identifiedusing any number of epitope mapping techniques, well known in the art.See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology,Vol. 66 (Glenn E. Morris, Ed., 1996). For example, linear epitopes maybe determined by e.g., concurrently synthesizing large numbers ofpeptides on solid supports, the peptides corresponding to portions ofthe protein molecule, and reacting the peptides with antibodies whilethe peptides are still attached to the supports. Such techniques areknown in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysenet al., 1984; Geysen et al., 1986. Similarly, conformational epitopesare readily identified by determining spatial conformation of aminoacids such as by, e.g., x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Methodsespecially applicable to the proteins of T. parva are fully described inPCT/US2004/022605 incorporated herein by reference in its entirety.

As discussed herein, the invention encompasses active fragments andvariants of the antigenic polypeptide. Thus, the term “immunogenic orantigenic polypeptide” further contemplates deletions, additions andsubstitutions to the sequence, so long as the polypeptide functions toproduce an immunological response as defined herein. The term“conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue. Inthis regard, particularly preferred substitutions will generally beconservative in nature, i.e., those substitutions that take place withina family of amino acids. For example, amino acids are generally dividedinto four families: (1) acidic—aspartate and glutamate; (2)basic—lysine, arginine, histidine; (3) non-polar—alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar—glycine, asparagine, glutamine, cystine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified as aromatic amino acids. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another hydrophobicresidue, or the substitution of one polar residue for another polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like; or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid that will not have a major effect on the biologicalactivity. Proteins having substantially the same amino acid sequence asthe reference molecule but possessing minor amino acid substitutionsthat do not substantially affect the immunogenicity of the protein are,therefore, within the definition of the reference polypeptide. All ofthe polypeptides produced by these modifications are included herein.The term “conservative variation” also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

The term “epitope” refers to the site on an antigen or hapten to whichspecific B cells and/or T cells respond. The term is also usedinterchangeably with “antigenic determinant” or “antigenic determinantsite”. Antibodies that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen.

An “immunological response” to a composition or vaccine is thedevelopment in the host of a cellular and/or antibody-mediated immuneresponse to a composition or vaccine of interest. Usually, an“immunological response” includes but is not limited to one or more ofthe following effects: the production of antibodies, B cells, helper Tcells, and/or cytotoxic T cells, directed specifically to an antigen orantigens included in the composition or vaccine of interest. Preferably,the host will display either a therapeutic or protective immunologicalresponse such that resistance to new infection will be enhanced and/orthe clinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms and/or clinicaldisease signs normally displayed by an infected host, a quicker recoverytime and/or a lowered viral titer in the infected host.

By “animal” is intended mammals, birds, and the like. Animal or host asused herein includes mammals and human. The animal may be selected fromthe group consisting of equine (e.g., horse), canine (e.g., dogs,wolves, foxes, coyotes, jackals), feline (e.g., lions, tigers, domesticcats, wild cats, other big cats, and other felines including cheetahsand lynx), ovine (e.g., sheep), bovine (e.g., cattle), bovine (e.g.,pig), avian (e.g., chicken, duck, goose, turkey, quail, pheasant,parrot, finches, hawk, crow, ostrich, emu and cassowary), primate (e.g.,prosimian, tarsier, monkey, gibbon, ape), ferrets, seals, and fish. Theterm “animal” also includes an individual animal in all stages ofdevelopment, including newborn, embryonic and fetal stages.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a”, “an”, and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicate otherwise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

Compositions

The present invention relates to a P. multocida vaccine or compositionwhich may comprise an attenuated P. multocida strain and apharmaceutically or veterinarily acceptable carrier, excipient, orvehicle, which elicits, induces or stimulates a response in an animal.

The term “nucleic acid” and “polynucleotide” refers to RNA or DNA thatis linear or branched, single or double stranded, or a hybrid thereof.The term also encompasses RNA/DNA hybrids. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs, uracyl, other sugars andlinking groups such as fluororibose and thiolate, and nucleotidebranches. The sequence of nucleotides may be further modified afterpolymerization, such as by conjugation, with a labeling component. Othertypes of modifications included in this definition are caps,substitution of one or more of the naturally occurring nucleotides withan analog, and introduction of means for attaching the polynucleotide toproteins, metal ions, labeling components, other polynucleotides orsolid support. The polynucleotides can be obtained by chemical synthesisor derived from a microorganism.

The term “gene” is used broadly to refer to any segment ofpolynucleotide associated with a biological function. Thus, genesinclude introns and exons as in genomic sequence, or just the codingsequences as in cDNAs and/or the regulatory sequences required for theirexpression. For example, gene also refers to a nucleic acid fragmentthat expresses mRNA or functional RNA, or encodes a specific protein,and which includes regulatory sequences.

An “isolated” biological component (such as a nucleic acid or protein ororganelle) refers to a component that has been substantially separatedor purified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinanttechnology as well as chemical synthesis.

The term “conservative variation” denotes the replacement of an aminoacid residue by another biologically similar residue, or the replacementof a nucleotide in a nucleic acid sequence such that the encoded aminoacid residue does not change or is another biologically similar residue.In this regard, particularly preferred substitutions will generally beconservative in nature, as described above.

The term “recombinant” means a polynucleotide with semisynthetic, orsynthetic origin which either does not occur in nature or is linked toanother polynucleotide in an arrangement not found in nature.

“Heterologous” means derived from a genetically distinct entity from therest of the entity to which it is being compared. For example, apolynucleotide may be placed by genetic engineering techniques into aplasmid or vector derived from a different source, and is a heterologouspolynucleotide. A promoter removed from its native coding sequence andoperatively linked to a coding sequence other than the native sequenceis a heterologous promoter.

The polynucleotides of the invention may comprise additional sequences,such as additional encoding sequences within the same transcriptionunit, controlling elements such as promoters, ribosome binding sites,5′UTR, 3′UTR, transcription terminators, polyadenylation sites,additional transcription units under control of the same or a differentpromoter, sequences that permit cloning, expression, homologousrecombination, and transformation of a host cell, and any such constructas may be desirable to provide embodiments of this invention.

Methods of Use and Article of Manufacture

The present invention includes the following method embodiments. In anembodiment, a method of vaccinating an animal comprising administering acomposition comprising an attenuated P. multocida strain and apharmaceutical or veterinarily acceptable carrier, excipient, or vehicleto an animal is disclosed. In one aspect of this embodiment, the animalis a bovine.

In one embodiment of the invention, a prime-boost regimen can beemployed, which is comprised of at least one primary administration andat least one booster administration using at least one commonpolypeptide, antigen, epitope or immunogen. Typically the immunologicalcomposition or vaccine used in primary administration is different innature from those used as a booster. However, it is noted that the samecomposition can be used as the primary administration and the boosteradministration. This administration protocol is called “prime-boost”.

A prime-boost regimen comprises at least one prime-administration and atleast one boost administration using at least one common polypeptideand/or variants or fragments thereof. The vaccine used inprime-administration may be different in nature from those used as alater booster vaccine. The prime-administration may comprise one or moreadministrations. Similarly, the boost administration may comprise one ormore administrations.

The dose volume of compositions for target species that are mammals,e.g., the dose volume of cow or bovine compositions, based on bacterialantigens, is generally between about 0.1 to about 2.0 ml, between about0.1 to about 1.0 ml, and between about 0.5 ml to about 1.0 ml.

The efficacy of the vaccines may be tested about 2 to 4 weeks after thelast immunization by challenging animals, such as bovines, with avirulent strain of P. multocida. Both homologous and heterologousstrains are used for challenge to test the efficacy of the vaccine. Theanimal may be challenged by IM or SC injection, spray, intra-nasally,intra-ocularly, intra-tracheally, and/or orally. Samples from joints,lungs, brain, and/or mouth may be collected before and post-challengeand may be analyzed for the presence of P. multocida-specific antibody.

The compositions comprising the attenuated bacterial strains of theinvention used in the prime-boost protocols are contained in apharmaceutically or veterinary acceptable vehicle, diluent or excipient.The protocols of the invention protect the animal from P. multocidaand/or prevent disease progression in an infected animal.

The various administrations are preferably carried out 1 to 6 weeksapart. Preferred time interval is 3 to 5 weeks, and optimally 4 weeksaccording to one embodiment, an annual booster is also envisioned. Theanimals, for example pigs, may be at least 3-4 weeks of age at the timeof the first administration.

It should be understood by one of skill in the art that the disclosureherein is provided by way of example and the present invention is notlimited thereto. From the disclosure herein and the knowledge in theart, the skilled artisan can determine the number of administrations,the administration route, and the doses to be used for each injectionprotocol, without any undue experimentation.

Another embodiment of the invention is a kit for performing a method ofeliciting or inducing an immunological or protective response against P.multocida in an animal comprising an attenuated P. multocidaimmunological composition or vaccine and instructions for performing themethod of delivery in an effective amount for eliciting an immuneresponse in the animal.

Another embodiment of the invention is a kit for performing a method ofinducing an immunological or protective response against P. multocida inan animal comprising a composition or vaccine comprising an attenuatedP. multocida strain of the invention, and instructions for performingthe method of delivery in an effective amount for eliciting an immuneresponse in the animal.

Yet another aspect of the present invention relates to a kit forprime-boost vaccination according to the present invention as describedabove. The kit may comprise at least two vials: a first vial containinga vaccine or composition for the prime-vaccination according to thepresent invention, and a second vial containing a vaccine or compositionfor the boost-vaccination according to the present invention. The kitmay advantageously contain additional first or second vials foradditional prime-vaccinations or additional boost-vaccinations.

The pharmaceutically or veterinarily acceptable carriers or vehicles orexcipients are well known to the one skilled in the art. For example, apharmaceutically or veterinarily acceptable carrier or vehicle orexcipient can be a 0.9% NaCl (e.g., saline) solution or a phosphatebuffer. Other pharmaceutically or veterinarily acceptable carrier orvehicle or excipients that can be used for methods of this inventioninclude, but are not limited to, poly-(L-glutamate) orpolyvinylpyrrolidone. The pharmaceutically or veterinarily acceptablecarrier or vehicle or excipients may be any compound or combination ofcompounds facilitating the administration of the vector (or proteinexpressed from an inventive vector in vitro); advantageously, thecarrier, vehicle or excipient may facilitate transfection and/or improvepreservation of the vector (or protein). Doses and dose volumes areherein discussed in the general description and can also be determinedby the skilled artisan from this disclosure read in conjunction with theknowledge in the art, without any undue experimentation.

The immunological compositions and vaccines according to the inventionmay comprise or consist essentially of one or more adjuvants. Suitableadjuvants for use in the practice of the present invention are (1)polymers of acrylic or methacrylic acid, maleic anhydride and alkenylderivative polymers, (2) immunostimulating sequences (ISS), such asoligodeoxyribonucleotide sequences having one or more non-methylated CpGunits (Klinman et al., 1996; WO98/16247), (3) an oil in water emulsion,such as the SPT emulsion described on page 147 of “Vaccine Design, TheSubunit and Adjuvant Approach” published by M. Powell, M. Newman, PlenumPress 1995, and the emulsion MF59 described on page 183 of the samework, (4) cationic lipids containing a quaternary ammonium salt, e.g.,DDA (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7)saponin or (8) other adjuvants discussed in any document cited andincorporated by reference into the instant application, or (9) anycombinations or mixtures thereof.

In an embodiment, adjuvants include those which promote improvedabsorption through mucosal linings. Some examples include MPL, LTK63,toxins, PLG microparticles and several others (Vajdy, M. Immunology andCell Biology (2004) 82, 617-627). In an embodiment, the adjuvant may bea chitosan (Van der Lubben et al. 2001; Patel et al. 2005; Majithiya etal. 2008; U.S. Pat. No. 5,980,912).

In an embodiment, the adjuvant may be inactivated bacteria, aninactivated virus, fractions of inactivated bacteria, bacteriallipopolysaccharides, bacterial toxins, or derivatives or combinationsthereof.

In an embodiment, the adjuvant comprises whole bacteria and/or viruses,including H. parasuis, clostridium, swine immunodeficiency virus (SIV),bovine circovirus (PCV), bovine reproductive and respiratory syndromevirus (PRRSV), Mannheimia, Pasteurella, Histophilus, Salmonella,Escherichia coli, or combinations and/or variations thereof. In severalembodiments, the adjuvant increases the animal's production of IgM, IgG,IgA, and/or combinations thereof.

REFERENCES

-   Rimler R B and Rhoades K R (1989). Pasteurella multocida. In: Adlam    C and Rutter J M (eds) Pasteurella and Pasteurellosis. London:    Academic Press, pp. 37-73.-   Rhoades K R and Rimler R B (1989). Fowl Cholera. London: Academic    Press-   Catry B, Chiers K, Schwarz S, Kehrenberg C, Decostere A and de Kruif    A (2005). Fatal peritonitis caused by Pasteurella multocida capsular    type F in calves. Journal of Clinical Microbiology 43: 1480-1483.-   Dungworth D C (1985). The respiratory system. In: Jubb K V F,    Kennedy P C and Palmer N (eds) Pathology of Domestic Animals,    Orlando, Fla.: Academic Press, pp. 448-489.-   Carter G R and de Alwis M C L (1989). Hemorrhagic septicemia. In:    Adlam C and Rutter J M (eds) Pasteurella and Pasteurellosis. London:    Academic Press, pp. 132-160.-   Shewen P E and Conlon J A R (1993). Pasteurella. In: Gyles C L and    Thoen C O (eds) Pathogenesis of Bacterial Infections in Animals.    Ames, Iowa: Iowa State University Press, pp. 216-225.-   Confer A W (1993). Immunogens of Pasteurella. Veterinary    Microbiology 37: 353-368.-   Confer A W, Nutt S H, Dabo S M, Panciera R J and Murphy G L (1996).    Antibody responses of cattle to outer membrane proteins of    Pasteurella multocida A:3. American Journal of Veterinary Research    57: 1453-1457-   Dalgleish R (1989) Studies on Experimental Pneumonia Pasteurellosis    in Calves. PhD thesis, University of Glasgow.-   Ewers C, Lubke-Becker A, Bethe A, Kiebling S, Filter M and Wieler L    H (2006). Virulence genotype of Pasteurella multocida strains    isolated from different hosts with various disease status.    Veterinary Microbiology 114: 304-317.-   Weekley L B, Veit H P, Eyre P (1998) Bovine pneumonic    pasteurellosis. Part I. Pathophysiology. Compendium on Continuing    Education for the Practicing Veterinarian, 20, S33eS46.-   Veterinary Laboratories Agency. (2007) VIDA 2007, Yearly Trends,    Cattle. http://www.defra.gov.uk/vla/reports/rep_vida07.htm.-   Frank G H (1989). Pasteurellosis of cattle. In: Adlam C and Rutter J    M (eds) Pasteurella and Pasteurellosis, London: Academic Press, pp.    197-222.-   Tatum F M, Tabatabai L B, Briggs R E. 2009. Sialic acid uptake is    necessary for virulence of Pasteurella multocida in turkeys. Microb    Pathog. 46(6):337-344.-   Steenbergen S M, Lichtensteiger C A, Caughlan R, Garfinkle J, Fuller    T E, Vimr E R. 2005. Sialic Acid metabolism and systemic    pasteurellosis. Infect Immun. 73(3):1284-1294.-   Briggs, R. E., Tatum, F. M. 2005. Generation and molecular    characterization of new temperature-sensitive plasmids intended for    genetic engineering of Pasteurellaceae. Appl Environ Micobiol    71:7187-7195.-   Chung J Y, Zhang Y, Adler B., 1998. The capsule biosynthetic locus    of Pasteurella multocida A:1. FEMS Microbiol Lett. 166(2):289-296.-   Lawrence, P. K., Shanthalingam, S., Dassanayake, R. P, Subramaniam,    R., Herndon, C. N., Knowles, D. P., Foreyt, W. J., Wayman, G.,    Marciel, A. M., Highlander, S. K., Srikumaran, S. 2010. Transmission    of Mannheimia haemolytica from domestic sheep (Ovis aries) to    bighorn sheep (Ovis canadensis): unequivocal demonstration with    green fluorescent protein-tagged organisms. Journal of Wildlife    Disease. 46 (3): 706-717.

The invention will now be further described by way of the followingnon-limiting examples.

EXAMPLES Example 1 Development of P. multocida hyaD Mutant Strain andCharacterization of Master Seed

A P. multocida mutant of strain 1062, unable to synthesize capsule(acapsular), was constructed by deleting a portion of the coding regionof hyaD and thereby inactivating the synthesis of glycosyl transferase(Chung et al., 1998). The acapsular mutant was created by following thepublished protocol (Briggs and Tatum 2005). The majority of the codingregions of P. multocida 1062 hyaD was obtained by PCR amplification,using the forward primer 5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ IDNO:14) and the reverse primer 5′-TGT AAT TTT CGT TCC CAA GGC-3′ (SEQ IDNO:15) (Briggs, R. E., Tatum, F. M. 2005). These two primers weresynthesized with an oligonucleotide synthesizer (Applied BiosystemsInc., CA) by Integrated DNA Technologies, Inc., Coralville, Iowa. ThePCR reactions were carried out using the GeneAmp LX PCR Kit (PE AppliedBiosystems, Foster City, Calif.) in a Perkin Elmer GeneAmp 9600thermocycler. Reaction conditions were 30 cycles, each consisting of 30seconds at 95° C., 45 seconds at 54° C., and 90 seconds at 72° C.

The PCR-generated hyaD fragment used here extended from the startingmethionine codon and ended 85 base pairs upstream of the stop codon. Thefragment was inserted into pCR2.1 (Invitrogen Inc., LaJolla, Calif.) andthe recombinant plasmid was electroporated into the E. coli strain DH11S(Life Technologies, Rockville, Md.) thus generating the plasmid,pCR2.1hyaDPm1062. This plasmid was isolated from E. coli by the alkalineSDS method and purified by CsCl centrifugation using standard methods.Both strands of the hyaD gene were sequenced using the Dye TerminatorChemistry kit from PE Applied Biosystems and samples were run on an ABIPrism 377 DNA Sequencer by the Nucleic Acids Facility, Iowa StateUniversity, Ames, Iowa.

The precise deletion within hyaD was produced by treating plasmid,pCR2.1hyaDPm1062 with the restriction enzyme BglII. This treatmentproduced a 225 bp deletion within hyaD which upon ligation resulted inan in-frame deletion. The deleted hyaD fragment was transferred into theEcoRI site within the multiple cloning site of plasmid pBCSK (StrategeneInc.) to produce pBCSKΔhyaDPm1062. Next, the Tn903 kanamycin resistanceelement (GenBlock) was inserted into the adjacent Sail site to producepBCSKΔhyaDPm1062kan^(R). Construction of the replacement plasmid wascompleted by ligating BssHII digested pBCSKΔhyaDPm1062kan^(R) to the 1.2kb temperature-sensitive origin of replication of plasmid, pCT109GA189(Briggs and Tatum, 2005). Because the ColE1 origin is inactive in P.multocida, only the ligation product generating plasmid,pCT109GA189ΔhyaDPm1062kan^(R), was capable of replicating within P.multocida strain 1062.

Replacement plasmid, pCT109GA189ΔhyaDPm1062kan^(R) was introduced intoP. multocida strain 1062 as follows. Cells were grown in Columbia brothto a density of OD₆₀₀=0.5. The cells were chilled on ice for 10 min andpelleted by centrifugation at 5000×g for 15 min. The cells wereresuspended in ice-cold distilled water and pelleted as described above.A second wash was done and the cell pellet was resuspended 1:3(bacteria:water) and placed on ice. The competent bacteria (100 μl) weremixed in a 0.1 cm electroporation cuvette (Bio-Rad) with the replacementplasmid ligation mixture. Immediately after adding DNA, the cells wereelectroporated (Gene pulser, Bio-Rad) at 18,000 V/cm, 800 ohm, and 25mFd, with resultant time constants ranging from 11 to 15 msec.

Chilled Columbia broth (1 ml) was added to the electroporated cells, andthe suspension was incubated at 25° C. for approximately 2 hours. Thecells were then plated onto Columbia blood agar plates containing 50μg/ml kanamycin. Colonies were visible after 24 hour incubation at 30°C. Colonies were transferred to 2 ml of Columbia broth containing 50μg/ml kanamycin and incubated overnight at 30° C. The next day,approximately 20 μl of the culture was spread onto dextrose starch agarplates containing 50 μg/ml kanamycin and incubated at 38° C., thenonpermissive temperature for the replacement plasmid. Cells possessingintegrated replacement plasmid survived antibiotic selection at thenon-permissive temperature for plasmid replication (38° C.). Thesesingle-crossover mutants could be easily identified phenotypicallybecause integration of replacement plasmid into hyaD of the hostresulted in loss of capsule. Wild-type capsular colonies of P. multocida1062 possess hyaluronic acid, the major capsule component, are mucoid inappearance and when viewed under obliquely transmitted light exhibit apearl-like iridescence. In contrast, the acapsular single-crossovermutants are non-mucoid and non-iridescent.

Several single-crossover mutants, possessing integrated replacementplasmid, were transferred to 5 ml Columbia broth without antibioticsupplementation and incubated at 30° C. overnight. The next day,approximately 2 μl of growth was transferred to fresh 5 ml Columbiabroth (without antibiotic) and incubated overnight at 30° C. Thisprocess was repeated several more times to allow for the resolution ofthe plasmid and mutant formation. After 5 such passages, cells weretransferred to dextrose-starch agar plates without supplementalantibiotic and incubated at 38° C. for 16 hours. The colonies whicharose on the non-selection plates consisted of both capsular andacapsular phenotypes. These results were expected; depending on wherereplacement-plasmid resolution occurred either wild-type or mutantcolonies were generated. The initial test to identify double crossovermutants (i.e. acapsular hyaD mutants) involved replica-plating acapsularcolonies onto dextrose starch agar plates with and without antibioticfollowed by overnight incubation at 38° C. Kanamycin sensitive acapsularcolonies were further analyzed by PCR using the hyaD primers describedpreviously. The PCR products of the putative hyaD mutants were comparedto those of the wild-type parent using agarose gel electrophoresis. PCRproducts that were of the expected size were sequenced using the hyaDF(5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ ID NO:14)) and hyaDR (5′-GTAATT TTC GTT CCC AAG GC-3′ (SEQ ID NO:15)) primers. Moreover, theputative hyaD mutants were analyzed by PCR for the absence oftemperature sensitive plasmid origin of replication of pCT109GA189 andfor the absence of the Tn903 kanamycin resistance element. A flowdiagram showing the construction of P. multocida 1062 hyaD mutant isshown in FIG. 1 (steps A-F).

A lyophilized culture of the mutant P. multocida 1062 (serotype A:3) wasobtained from NADC, Ames Iowa. The lyophilized powder was rehydratedusing sterile distilled water and streaked on TSA plates supplementedwith 5% sheep blood and incubated at 37° C. overnight. The next day,three individual colonies were picked and colony PCR was performed usinghyaDF and hyaDR primers as described earlier (Lawrence et al., 2010).The clones as expected, amplified a band at 2.691 kb, when compared tothe parent strain (2.916 kb), indicating a truncated hyaD gene. A singlecolony from one of the clones was inoculated into autoclaved brain heartinfusion (BHI) medium and incubated at 37° C./200 rpm overnight. Thenext day, cultures were diluted to 0.4 OD (optical density at 600 nm) inBHI broth and allowed to grow until log phase. Once the cultures reachedlog phase (OD₆₀₀=0.8), an equal volume of 50% sterile glycerol wasadded, mixed and aliquoted into 2 ml cryovials. The master seed wasfrozen at a concentration of 2.6×10⁹ CFU/ml. The vials were labeled asP. multocida 1062 hyaDΔ-4PKL and stored at −70° C. at NewportLaboratories Research and Development, located at 1810 Oxford Street,Worthington, Mn-56187.

The master seed stock was seeded onto TSA plates supplemented with 5%sheep blood, and a single colony was picked to confirm the presence oftruncated hyaD by PCR (FIG. 2). The master seed was sent to internalquality control (QC) laboratory for sterility testing according to 9CFR113.27(b), and was devoid of any extraneous (fungal) growth. A TSA platecontaining P. multocida 1062 hyaDΔ-1PKL was obtained from the QClaboratory and sent to internal diagnostics laboratory foridentification/confirmation according to 9CFR 113.64(c)4. Afterconfirming that the growth obtained from the QC laboratory was mutant P.multocida 1062 hyaDΔ-1PKL, we analyzed a single colony from the TSAplate again for the presence of truncated hyaD gene by PCR. The masterseed was tested for safety in outbred mice [Hsd:ICR (CD-1®), Harlan],per 9CFR 113.33 and was found to be safe.

The master seed stock was seeded onto TSA plates supplemented with 5%sheep blood, and a single colony was picked and colony PCR was performedas described earlier (Lawrence et al., 2010) using hyaDF and hyaDRprimers. The agarose gel image is shown in FIG. Lanes: 1—molecularweight marker; 2—PCR product of P. multocida 1062 hyaDΔ-4PKL showingtruncated hyaD gene (2.691 kb); 3—PCR product of P. multocida 1062 wildtype showing full length hyaD gene (2.916 kb).

Example 2 nanPU Mutant Strain Development and Master SeedCharacterization

A P. multocida mutant of strain 1062, unable to add sialic acid residuesto lipooligosaccharide (LOS) molecules, was constructed by deleting aportion of the coding region of nanPU and thereby inactivating thesynthesis of a sialic acid transferase enzyme complex (Tatum et al.,2009; Steenbergen et al., 2005). The locus nanP encodes a sialicacid-binding periplasmic protein SiaP and the locus nanU encodes sialicacid TRAP transporter permease protein SiaT-23. The sialic aciddeficient mutant was created by following the published protocol (Briggsand Tatum 2005). The coding regions of nanP and nanU of P. multocida1062 were obtained by PCR amplification, using the forward and reverseprimers 1062nanPU_F 5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′ (SEQ IDNO:6)/1062nanPU_delR 5′-GTC ACA CCT TGA CTT TTG AAG AAT TCA-3′ (SEQ IDNO:7); and 1062nanPU_delF 5′-AAT TCC AAT TGC GGT TCA CTT TGG CA-3′ (SEQID NO:8)/1062nanPU_R 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA-3′ (SEQ IDNO:9), respectively (FIG. 3A). The PCR reactions were carried out usingthe GeneAmp LX PCR Kit (PE Applied Biosystems, Foster City, Calif.) in aPerkin Elmer GeneAmp 9600 thermocycler. Reaction conditions were 30cycles, each consisting of 30 seconds at 95° C., 45 seconds at 54° C.,and 90 seconds at 72° C.

The PCR-generated nanP and nanU fragments were digested with EcoRI andMunI respectively, ligated and cloned into PCR2.1 vector (InvitrogenInc., LaJolla, Calif.). The recombinant plasmid was electroporated intothe E. coli strain DH11S (Life Technologies, Rockville, Md.) thusgenerating the plasmid, pCR2.1nanPUPm1062. This plasmid was isolatedfrom E. coli by the alkaline SDS method and purified by CsClcentrifugation using standard methods. Both strands of the nanPU genewere sequenced using the Dye Terminator Chemistry kit from PE AppliedBiosystems and samples were run on an ABI Prism 377 DNA Sequencer by theNucleic Acids Facility, Iowa State University, Ames, Iowa.

The deleted nanPU fragment was transferred into the EcoRI site withinthe multiple cloning site of plasmid pBCSK (Strategene Inc.,) to producepBCSKΔnanPUPm1062 (FIG. 1B). Next, the Tn903 kanamycin resistanceelement (GenBlock) was inserted into the adjacent SalI site to producepBCSKΔnanPUPm1062kanR. Construction of the replacement plasmid wascompleted by ligating BssHII digested pBCSKΔnanPUPm1062kanR to the 1.2kb temperature-sensitive origin of replication of plasmid, pCT109GA1894(Briggs and Tatum, 2005). Because the ColE1 origin is inactive in P.multocida, only the ligation product generating plasmid,pCT109GA189ΔnanPUPm1062kanR, was capable of replicating within P.multocida strain 1062 (FIGS. 3C & D).

Replacement plasmid, pCT109GA189ΔnanPUPm1062kanR was introduced into P.multocida strain 1062 as follows. Cells were grown in Columbia broth toa density of OD600=0.5. The cells were chilled on ice for 10 min andpelleted by centrifugation at 5000×g for 15 min. The cells wereresuspended in ice-cold distilled water and pelleted as described above.A second wash was done and the cell pellet was resuspended 1:3(bacteria:water) and placed on ice. The competent bacteria (100 μl) weremixed in a 0.1 cm electroporation cuvette (Bio-Rad) with the replacementplasmid ligation mixture. Immediately after adding DNA, the cells wereelectroporated (Gene pulser, Bio-Rad) at 18,000 V/cm, 800 ohm, and 25mFd, with resultant time constants ranging from 11 to 15 msec.

Chilled Columbia broth (1 ml) was added to the electroporated cells, andthe suspension was incubated at 25° C. for approximately 2 hours. Thecells were then plated onto Columbia blood agar plates containing 50μg/ml kanamycin. Colonies were visible after 24 hour incubation at 30°C. Colonies were transferred to 2 ml of Columbia broth containing 50μg/ml kanamycin and incubated overnight at 30° C. The next day,approximately 20 μl of the culture was spread onto dextrose starch agarplates containing 50 μg/ml kanamycin and incubated at 38° C., thenonpermissive temperature for the replacement plasmid. Cells possessingintegrated replacement plasmid survived antibiotic selection at thenon-permissive temperature for plasmid replication (38° C.).

Several single-crossover mutants, possessing integrated replacementplasmid, were transferred to 5 ml Columbia broth without antibioticsupplementation and incubated at 30° C. overnight. The next day,approximately 2 μl of growth was transferred to fresh 5 ml Columbiabroth (without antibiotic) and incubated overnight at 30° C. Thisprocess was repeated several more times to allow for the resolution ofthe plasmid and mutant formation. After 5 such passages, cells weretransferred to dextrose-starch agar plates without supplementalantibiotic and incubated at 38° C. for 16 hours. The colonies whicharose on the non-selection plates consisted of both wild type and mutantphenotypes. These results were expected; depending on wherereplacement-plasmid resolution occur either wild-type or mutant colonieswere generated. The initial test to identify double crossover mutants(i.e. nanPU mutants) involved replica-plating colonies onto dextrosestarch agar plates with and without antibiotic followed by overnightincubation at 38° C. Kanamycin sensitive colonies were further analyzedby PCR using the nanPU primers described previously. The PCR products ofthe putative nanPU mutants were compared to those of the wild-typeparent using agarose gel electrophoresis. PCR products that were of theexpected size were sequenced using the nanPUF (5′-TTC CCT AGC TCA CAGTTA GGT GAT-3′) (SEQ ID NO:6) and nanPUR (5′-TCT GCA ATT TCT TTC CAT TCTTTT GGA TCT-3′) (SEQ ID NO:16) primers. Also the putative nanPU mutantswere sequenced, analyzed by PCR for the absence of temperature sensitiveplasmid origin of replication of pCT109GA189 and for the absence of theTn903 kanamycin resistance element. The nanPU mutants were assayed foruptake of sialic acid from the culture media using the thiobarbituricacid assay 5. A flow diagram showing the construction of P. multocida1062 nanPU mutant is shown in FIG. 3 (Steps A-F).

A blood agar plate culture of the mutant P. multocida 1062 (serotypeA:3) nanPU mutant was obtained from NADC, Ames Iowa on 4 May 2012. Thelyophilized powder was rehydrated using sterile distilled water andstreaked on TSA plates supplemented with 5% sheep blood and incubated at37° C. overnight. The next day, three individual colonies were pickedand colony PCR was performed using nanPUF 5′-TTC CCT AGC TCA CAG TTA GGTGAT-3′ (SEQ ID NO:6) and nanPUR 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGATCT-3′ (SEQ ID NO:16) primers as described earlier (Lawrence et al.,2010). The clones as expected, amplified a band at 1.3 kb, when comparedto the parent strain (3.150 kb), indicating a truncated nanPU gene. Asingle colony from one of the clones was inoculated into autoclavedbrain heart infusion (BHI) medium and incubated at 37° C./200 rpmovernight. The next day, cultures were diluted to 0.4 OD (opticaldensity at 600 nm) in BHI broth and allowed to grow until log phase.Once the cultures reached log phase (OD600=0.8), an equal volume of 50%sterile glycerol was added, mixed and aliquoted into 2 ml cryovials(frozen at 6.9×10⁸ CFU/ml).

The master seed stock was seeded onto TSA plates supplemented with 5%sheep blood, and a single colony was picked to confirm the presence oftruncated nanPU by PCR (FIG. 4). The master seed was sent to internalquality control (QC) laboratory for sterility testing according to 9CFR113.27(b), and was devoid of any extraneous (fungal) growth. The masterseed was tested for safety in outbred mice [Hsd:ICR (CD-1®), Harlan],per 9CFR 113.33 and was found to have adverse effect.

The master seed stock was seeded onto TSA plates supplemented with 5%sheep blood, and a single colony was picked and colony PCR was performedas described earlier using nanPUF and nanPUR primers. FIG. 4 shows theimage of an agarose gel: 1—MW marker; 2—PCR product of P. multocida 1062P. mult. 1062 Nan showing truncated nanPU gene (1.3 Kb); 3—PCR productof P. multocida 1062 wild type showing full length nanPU gene (3.150Kb); negative control lacking template DNA.

Example 3 Development of P. multocida nanPU/hyaD Double Knockout Mutant

To develop a double knockout mutant of P. multocida 1062 A:3, a singledeletion mutant in hyaD was constructed as described in section IIa andwas used as a base to knock out nanPU locus as described in section IIb.

A blood agar plate of mutant P. multocida 1062 (serotype A:3) nanPU/hyaDwas obtained from NADC, Ames Iowa on 4 May 2012. The lyophilized powderwas rehydrated using sterile distilled water and streaked on TSA platessupplemented with 5% sheep blood and incubated at 37° C. overnight. Thenext day, three individual colonies were picked and colony PCR wasperformed using nanPUF 5′-TTC CCT AGC TCA CAG TTA GGT GAT-3′ (SEQ IDNO:6)/nanPUR 5′-TCT GCA ATT TCT TTC CAT TCT TTT GGA TCT-3′ (SEQ IDNO:16) and hyaDF 5′-ATG ATA TTT GAG AAG TCG GCG G-3′ (SEQ IDNO:14)/hyaDR 5′-GTA ATT TTC GTT CCC AAG GC-3′ (SEQ ID NO:17) primers, asdescribed earlier (Lawrence et al., 2010). The clones as expected,amplified a band at 1.3 kb, when compared to the parent strain (3.150kb), indicating a truncated nanPU gene. A single colony from one of theclones was inoculated into autoclaved brain heart infusion (BHI) mediumand incubated at 37° C./200 rpm overnight. The next day, cultures werediluted to 0.4 OD (optical density at 600 nm) in BHI broth and allowedto grow until log phase. Once the cultures reached log phase(OD600=0.8), an equal volume of 50% sterile glycerol was added, mixedand aliquoted into 2 ml cryovials. The master seed was frozen at aconcentration of 2.64×10⁸ CFU/ml.

The master seed stock was seeded onto TSA plates supplemented with 5%sheep blood, and a single colony was picked to confirm the presence oftruncated nanPU and hyaD by PCR (FIG. 5). The master seed was sent tointernal quality control (QC) laboratory for sterility testing accordingto 9CFR 113.27(b), and was devoid of any extraneous (fungal/bacterial)growth. A TSA plate containing P. mult 1062 Nan-Hya-May 8, 2012 wasobtained from the QC laboratory and sent to internal diagnosticslaboratory for identification/confirmation according to 9CFR 113.64(c)4.After confirming that the growth obtained from the QC laboratory wasmutant P. mult 1062 Nan-Hya-May 9, 2012, we analyzed a single colonyfrom the TSA plate again for the presence of truncated nanPU and hyaDgene by PCR. The master seed was tested for safety in outbred mice[Hsd:ICR (CD-1®), Harlan], per 9CFR 113.33 and was found to have noadverse effect.

III. Evaluate the Efficacy of Pasteurella multocida Vaccine Candidates

Objective:

-   1. Vaccinate calves with modified live P. multocida vaccine through    subcutaneous route.-   2. Challenge with P. multocida 1062 wild type to determine vaccine    efficacy.    Materials and Methods:-   Product: Log phase cultures of P. multocida hyaD, P. multocida    hyaD/nanPU and P. multocida nanPU mutants.-   Animals and Housing: There were a total of 15 calves, 4 weeks of age    and housed in 4 different pens as described in Table 1.

TABLE 1 Treatment Groups Total Dose/CFU Group Treatment per animalRoute/volume Calf Id #. 1 P. multocida hyaD  1.14 × 10⁹ subcutaneous115, 114, 117 2 ml 2 P. multocida  1.02 × 10⁹ subcutaneous 99, 88, 112hyaD/nanPU 2 ml 3 P. multocida nanPU 1.226 × 10⁹ subcutaneous 110, 105,91 2 ml 4 Control RPMI subcutaneous 119, 89, 97, 95, medium 2 ml 94, 101Vaccination:

-   -   1. A fresh glycerol stock of P. multocida vaccine was grown        overnight in BHI medium, plated (TSA) the next day and incubated        at 37° C. The following day plates were scraped and diluted into        RPMI medium supplemented with 2% inactivated fetal bovine serum.        The inoculum was grown at 37° C./200 rpm until desired OD₆₀₀ was        achieved.    -   2. The culture was diluted to 10⁹ CFU/vaccine dose and dilution        plated to enumerate the exact CFU/ml the following day.    -   3. The vaccine was transported on ice and kept on ice during        vaccination.    -   4. Route and dose: subcutaneous, 1 ml per each side of neck.    -   5. The injection site was observed for adverse reaction.    -   6. The calves in the control group received RPMI medium only.

-   The study schedule is described in Table 2.    Challenge: P. multocida 1062 Wild Type    -   1. A fresh glycerol stock of P. multocida 1062 was grown 0/N in        BHI medium, plated (TSA) the next day and incubated at 37° C.        The following day plates were scraped and diluted into RPMI        medium supplemented with 2% inactivated fetal bovine serum. The        inoculum was grown at 37° C./200 rpm until desired OD₆₀₀ was        achieved.    -   2. The culture was diluted to approximately 10¹⁰ CFU/challenge        dose and dilution plated to enumerate the exact CFU/ml the        following day.    -   3. The inoculum was transported on ice and kept on ice during        challenge.    -   4. Route: Trans-tracheal using 14G×1 inch needle.    -   5. Dose: 3.78×10¹⁰ CFU/animal in 20 ml RPMI, chased with 60 ml        RPMI.

-   Once completed the remaining inoculum was immediately dilution    plated in the lab.

-   The calves were monitored for change in behavior including lethargy,    coughing, nasal discharge and scored as shown in Table 2. Rectal    temperatures were monitored for calves showing clinical signs.    Necropsy Directions:    -   1. Animals that are dead, weak or showing clinical signs of        pneumonia were euthanized and necropsied immediately by a        licensed veterinarian.    -   2. The remaining calves were euthanized humanely on day 5 by        injecting pentobarbital (Euthasol, 20-30 ml at the discretion of        the assigned veterinarian) per animal.    -   3. The lungs were scored for pneumonic lesions and recorded as        percent lesion on each lobe.    -   4. The lung tissues were collected for histopathology.    -   5. Swabs were taken from lungs (lesions) and trachea for the        recovery of challenge organism.

TABLE 2 Study Schedule Age Event 4 weeks old Day 0—bleed, swab andvaccinate 7 days post vaccination bleed and swab 18 days postvaccinate—bleed and swab & challenge with P. multocida 1062 wild typeObserve clinical signs starting the day of challenge, euthanize anycalves if necessary. Euthanize and necropsy all on day 4 post challenge*The calves were observed for feed intake and rectal temperatures takenmorning and evening post challenge.

TABLE 3 Clinical signs for scoring Criteria for Post ChallengeObservations 0 = Normal 1 = Depression, Anorexia, Cough, NasalDischarge, Dyspnea 2 = Severely Depressed, Unable to Rise or Walk,Euthanized for Humane Reasons 3 = Dead On Arrival (DOA)Results:

None of the vaccinated calves showed any injection site swelling,granuloma or anaphylaxis. One day post challenge calf#97 died. Theremaining calves in the control group were depressed lethargic andanorexic. The vaccinates and control calves were euthanized on day 4,post challenge. The lungs from the control calves showed severe purulentbronchopneumonia with mild dysplasia of terminal bronchiole epithelium.The control group had an average lung lesion score of 43.26%. Thevaccinates on the other hand showed no febrile response. Upon necropsythe lungs from vaccinates showed mild purulent lesions, moderateendobronchial polyps and prior viral infection. Overall all the threevaccine candidates tested significantly reduced the lung lesion whencompared to non-vaccinates and can be used as vaccine when injectedsubcutaneously. However, among the three vaccine candidates tested P.multocida nanPU was more potent in reducing the lung lesion (averagelung lesion score 10.2%) compared to P. multocida hyaD, (average lunglesion score 17.735%) or P. multocida hyaD/nanPU (average lung lesionscore 13.97%).

* * *

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A method of vaccinating an animal comprisingadministering a vaccine composition comprising an attenuated Pasteurellamultocida (P. multocida) strain that provides a protective immuneresponse in a bovine against P. multocida, or diseases caused by P.multocida; wherein the attenuated strain comprises deletions or partialdeletions in its hyaD gene and its nanPU gene, relative to the virulentparental strain from which the attenuated strain was produced; andwherein the nanPU gene present in the attenuated P. multocida strainencodes a polypeptide having the sequence set forth in SEQ ID NO:13. 2.The method of claim 1, wherein the bovine is from about 4 weeks old to 6weeks old.
 3. The method of claim 1, wherein the parental strain hyaDgene comprises the sequence set forth in SEQ ID NO: 1and the parentalstrain nanPU gene comprises the sequence as set forth in SEQ ID NO: 5.4. The method of claim 3, wherein the parental strain hyaD gene consistsof the sequence set forth in SEQ ID NO: 1 and the parental strain nanPUgene consists of the sequence as set forth in SEQ ID NO:
 5. 5. Themethod of claim 1, wherein the parental strain hyaD gene encodes aprotein having the sequence set forth in SEQ ID NO: 2 and the parentalstrain nanPU gene encodes proteins having the sequences as set forth inSEQ ID NOs:10 and
 11. 6. The method of claim 1, wherein the attenuatedstrain expresses level(s) of HyaD and/or NanP, NanU peptide(s) that aresignificantly reduced or undetectable, relative to the attenuatedstrain's corresponding virulent parental strain.
 7. The method of claim1, wherein the attenuated strain has mutations in the same genes,relative to its corresponding virulent parental strain, as does theattenuated strain deposited at the ATCC under the Patent DepositDesignation PTA-120624.
 8. The method of claim 1, wherein the attenuatedstrain is the strain deposited at the ATCC under the Patent DepositDesignation PTA-120624.
 9. The method of claim 1, wherein the attenuatedstrain has a deletion in its hyaD gene such that the attenuated strainproduces a truncated protein having the sequence as set forth in SEQ IDNO:
 4. 10. The method of claim 1, wherein the attenuated strain hasdeletions in both its hyaD and nanPU genes, such that the attenuatedstrain does not produce wild type HyaD, NanP and NanU proteins, as doesits corresponding parental strain, but instead produces truncatedproteins having the sequences as set forth in SEQ ID NOs: 4 and
 13. 11.The method of claim 1, wherein the vaccine composition further comprisesa pharmaceutically or veterinary acceptable vehicle, diluent orexcipient, and an adjuvant.
 12. The method of claim 11, wherein theadjuvant is inactivated bacteria, inactivated virus, fractions ofinactivated bacteria, bacterial lipopolysaccharides, bacterial toxins,or combinations thereof.
 13. The method of claim 1, wherein thecomposition further comprises an additional antigen(s) associated with abovine pathogen other than P. multocida.
 14. The method of claim 13,wherein the additional antigen(s) elicits in a bovine an immune responseagainst bovine respiratory disease (BRD).